Filamentous fungi can produce high yields of proteins and metabolites. Impressive increases in the secretion of homologous proteins were obtained with traditional strain-improvement strategies based on various mutagenesis approaches. As such, industrial strains have been created that secrete >20 g/l of a specific endogenous protein. In this way, filamentous fungi seem promising organisms for the production of heterologous proteins of biomedical interest (Maras et al., 1999; Punt et al., 2002).
However, unlike mammalian cells, these lower eukaryotic organisms do not synthesize complex type protein-linked oligosaccharides. This inability hampers the use of therapeutic glycoproteins produced by filamentous fungi, since they mostly synthesize high-mannose type N-glycans. Due to the presence of several lectins on human cells, glycoproteins carrying this type of glycosylation are rapidly cleared from the blood stream. This significantly reduces their therapeutic value.
Not only are lower eukaryotes like filamentous fungi, unable to synthesize complex type oligosaccharides, they sometimes also elongate the high-mannose type glycans with fungal-specific glycan residues like mannosephosphate, α-1,3-mannose and galactofuranose. Some of these residues induce an immunogenic response in humans, again reducing the therapeutic value of such glycoproteins.
Protein N-glycosylation originates in the endoplasmic reticulum (ER), where an N-linked oligosaccharide (Glc3Man9GlcNAc2) assembled on dolichol (a lipid carrier intermediate) is transferred to the appropriate Asn of a nascent protein. This is a co-translational event common to all eukaryotic organisms. The three glucose residues and one specific α-1,2-linked mannose residue are removed by specific glucosidases and an α-1,2-mannosidase in the ER, resulting in the core oligosaccharide structure, Man8GlcNAc2. Proteins with this core sugar structure are transported to the Golgi apparatus where the sugar moiety undergoes various modifications. Significant differences exist in the modifications of the sugar chain in the Golgi apparatus between lower and higher eukaryotes.
In mammalian cells, the modification of the sugar chain can follow three different pathways depending on the protein moiety to which it is added. That is: (1) the core sugar chain does not change; (2) the core sugar chain is changed by adding the N-acetylglucosamine-1-phosphate moiety (GlcNAc-1-P) in UDP-N-acetyl glucosamine (UDP-GlcNAc) to the 6-position of mannose in the core sugar chain, followed by removal of the GlcNAc moiety to form an acidic sugar chain in the glycoprotein; and (3) the core sugar chain is first converted into Man5GlcNAc2 by removing three mannose residues with Golgi a Mannosidase I; Man5GlcNAc2 is then further modified by adding GlcNAc and removing two more mannose residues, followed by sequentially adding GlcNAc, galactose (Gal), and N-acetylneuraminic acid (also called sialic acid (NeuNAc)) to form various hybrid or complex sugar chains (R. Kornfeld and S. Kornfeld, 1985; Chiba et al., 1998).
In filamentous fungi like Trichoderma reesei, only a part of the Man8(9)GlcNAc2 structures are (partially) trimmed down to Man5GlcNAc2. These oligosaccharides can then be further modified to fungal-specific glycans through the addition of mannosephosphate residues in a diester linkage. As such, a variety of sugar residues can be found on Trichoderma secreted glycoproteins, consisting of Man5-8(9)GlcNAc2 with or without one or two mannosephosphate residues. An exception to this general Trichoderma glycosylation pattern is the Rut-C30 strain, producing mainly GlcMan7(9)GlcNAc2 or GlcMan7(9)GlcNAc2-P-Man (Maras et al., 1997).
A clear need exists for a fungal strain, such as a Trichoderma strain, that is able to secrete large amounts of a heterologous protein with a more human-compatible glycosylation profile. As such, the Rut-C30 strain of T. Reesei which is a hypersecretor of endogenous cellulases (up to 30 g/l), would be an interesting strain for heterologous protein production, but it is hampered by its aberrant glycosylation pattern, compared to the wild-type Qm6a strain and to most of the industrial mutant strains. In these Trichoderma strains, a first α-1,2-linked glucose residue is removed by glucosidase I, after transfer of the Glc3Man9GlcNAc2 structure to the protein. This is followed by the removal of the two α-1,3-linked glucose residues by glucosidase II. However in the Rut-C30 strain, NMR analysis revealed that more than 80% of the glycan structures synthesized on cellobiohydrolase I (CBH I) still contained one α-1,3-linked glucose residue at the end of the α-1,3-arm of the high-mannose core structure (Maras et al., 1997). This indicates a malfunction at the level of the glucosidase II. This malfunction could be due to a reduced expression level of the enzyme.